expressions of GS and LDH-1 in LGP and FC after manganese treatment
3. Assembling the sandwich(es)
4.2.5.4 Staining methods
To confirm that the transfer of the proteins to the membrane occurred, a total protein stain such as Ponceau S is most commonly used. It is rapid, reversible, and does not interfere with subsequent immunodetection. In addition, staining the acrylamide gel after transfer confirms the complete migration of the proteins out of the gel. Beside Ponceau S, Coomassie Brilliant Blue R-250 is commonly used for the detection of proteins, and is sensitive in a range of 0.5 to 25 µg of protein. Within this range, it also follows the Beer-Lambert law and thus can be quantitative as well as qualitative.
4.2.5.4.1 Coomassie Brilliant Blue R-250:
1. Soak blott (after dd H2O rinse) in methanol.
2. Stain PVDF membrane with 0.1% Coomassie R-250 in 40% MeOH for no longer than ONE MINUTE - usually 15 to 20 seconds are sufficient. (Staining for longer periods of time will result in high background and will interfere with extraction and cleavage).
3. Destain with 50% methanol, several changes. 4. Rinse extensively with dd H2O.
5. Cut out the band of interest.
4.2.5.4.2 Ponceau S:
1. Rinse the PVDF membrane with methanol for 10 minutes. 2. Wash the PVDF membrane with dd H2O three times.
3. Stain the PVDF membrane in 0.25% Ponceau S in 1% Acetic acid for 10 minutes (until protein bands are visible).
4. Rinse the membrane in dd H2O three times.
5. Dry PVDF membrane on 3M paper.
4.2.6 Immunohistochemistry
4.2.6.1 Introduction
Immunohistochemistry is the localization of antigens (proteins) in tissue sections by the use of labelled antibodies as specific reagents through antigen-antibody interactions that are
visualized by a marker such as fluorescent dye, enzymes, radioactive compounds or colloidal gold.
Albert H. Coons and his colleagues (Coons et al. 1955) were the first to label antibodies with a fluorescent dye, and use it to identify antigens in tissue sections. With the expansion and development of immunohistochemistry technique, enzyme labels have been introduced such as peroxidase (Nakane and Pierce, 1967; Avrameas and Uriel, 1966) and alkaline phosphatase (Mason and Sammons, 1978). Colloidal gold (Faulk and Taylor, 1971) label has also been discovered and used to identify immunohistochemical reactions at both light and electron microscopy level. Other labels include radioactive elements, and the immunoreaction can be visualized by autoradiography. Because immunohistochemistry involves a specific antigen-antibody reaction, it has apparent advantages over traditionally used special enzyme staining techniques that identify only a limited number of proteins, enzymes and tissue structures. Therefore, immunohistochemistry has become a crucial technique and is widely used in many medical research laboratories as well as clinical diagnostics. There are numerous immunohistochemistry methods that may be used to localize antigens. The selection of a suitable method should be based on parameters such as the type of specimen under investigation and the degree of sensitivity required.
Direct method: The direct method is a “one step staining method”, and involves a labelled
antibody reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is fast. However, it is insensitive due to low signal amplification and is, therefore, rarely used after the indirect method has been developed.
Indirect method: The indirect method involves the use of an unlabelled primary antibody
(first layer) which reacts with the antigen in the tissue, and a labelled secondary antibody (second layer) which reacts with the primary antibody (Note: the secondary antibody must be directed against the IgG of the animal species in which the primary antibody has been raised). This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. In addition, it has also economical advantages since one labelled second layer antibody can be used with several first layer antibodies to different antigens. The second layer antibody can be labelled with a fluorescent dye such as FITC, rhodamine or Texas Red, and this is called indirect
immunofluorescence method. The second layer antibody may be labelled with an enzyme
such as peroxidase, alkaline phosphatase or glucose oxidase, and this is called indirect
Controls: Special controls must be run in order to test the protocols and for the specificity of
the antibody being used.
Negative control is to test for the specificity of the antibody. First, no staining must be shown after omission of the primary antibody or the replacement of the specific primary antibody by a normal serum (must be the same species as the primary antibody).
Using immunohistochemistry, we studied the localization of two enzymes, i.e. glutamine synthetase (GS) and lactate dehydrogenase (LDH-1) in different regions of rat brains, in particular, the frontal cortex and the globus pallidus after intraperitoneally injection of 100µM MnCl2/day for 4 consecutive days as a sub-acute model of manganese neurotoxicity.
4.2.6.2 Experimental procedures
Chemicals Materials
MnCl2 Pipettes (20, 100, 200, 1000 µl)
Pentobarbital Centrifuge tube (50 ml) Phosphate buffered saline Vibratome
Formaldehyde Forceps Ethanol Scissor
H2O2 Syringe and needle
1X PBS (0.1 M, pH 7.5)
Sprague Dawley rats (150-200 g) were injected intraperitoneally with MnCl2 (50
mg/kg/day) on 4 consecutive days. On day 5, the rats were anesthetized with pentobarbital (Somnotol®, 65 mg/kg) which was administered intraperitoneally. The animals were perfused through the heart (see “Perfusion” Fig. 4.2) with a solution of 0.1 M phosphate buffered saline (PBS, pH 7.5) followed by 10% formaldehyde in 0.1 M phosphate buffer (PBS, pH 7.5) (“formaline”). The rats were decapitated; brains were removed and placed in 10% paraformaldehyde. After 24 hours, the formaldehyde was replaced by 1X PBS, and stored at 4°C. The brains of the animals were cutted sagittally in 40 µm slices with a vibratome. All sections were stored in borate buffered saline (pH 8.4) at 4°C for subsequent staining.
Perfusion through the heart
1. Aneasthetize rats by intraperitoneal somnotol injection (65 mg/kg). 2. Place the rats on their backside on ice.
3. Swab abdomen with 70 % ethanol. Cut horizon between abdomen and the chest; be carefully not to cut other organs.
4. Open the chest chamber and fix it. 5. Cut the diaphragm to expose the heart. 6. Fix the heart with a forcep.
7. Insert carefully the needle into the left ventricle (see the pulse in syringe). 8. Fix the needle with a long forcep.
9. Cut the aorta.
10. Inject 0.1 M phosphate buffered saline (PBS, pH 7.5) three times each 50 ml. 11. Inject 10% formaldehyde (formaline phosphate) three times each 50 ml. 12. Decapitate the rats and remove the brains.
13. Put the brains into 10% formaldehyde and store them at 4°C.
14. Replace the medium after 24 hours by 0.1 M phosphate buffered saline and store at 4°C.
Figure 4.1: Perfusion through the heart
Schematic representation of perfusion through the heart. The injection was carried out through the left venrticle, while the aorta is cut at the same time.
Sections were placed in 24 multiwell plates and washed for 10 minutes at room temperature with phosphate buffered saline (1X PBS) and 0.3% hydrogen peroxide (H2O2) to
oxidize the cellular peroxidase enzyme.Sections were then washed three times with 1X PBS for each 10 minutes to remove the excess of H2O2. Afterwards, the sections were incubated for
20 minutes with a solution containing 1X PBS, 0.5 % Triton X100, and 5% donkey serum to block the sections. Subsequently, the sections were incubated for 24 hours at 4°C with the
L e ft v e n tric le R ig h t v e n tric le A o rta Right ventricle Aorta Left ventricle Cutting site L e ft v e n tric le R ig h t v e n tric le A o rta Right ventricle Aorta Left ventricle Cutting site
4.2.6.3 Staining process
Chemicals Materials 0.3% H2O2 24 Multiwell plates 1X PBS (0.1 M, pH 7.5) Microscope slides0.5 % Triton X100 Microscope cover glass
5% donkey serum Shaker
Primary antibodies (GS and LDH-1) Fine brush
Secondary antibody (GS and LDH-1) Culture dishes
3,3-diaminobenzidine (DAB) 0.05% Parafilm
Nickelammoniumsulfate (NH4)2Ni(SO4)2
Ethanol (75%, 95%, and 100%)
Xyelene Permount solution (Touene Solution)
primary antibody (1:500 for GS, and 1:250 for LDH-1), 5% donkey serum, 0.5% Triton X100 in 1X PBS. Primary antibodies included the monoclonal antibody (directed against GS, and LDH-1). At the next day, sections were washed 3 times for 10 minutes in 1X PBS, and then transferred to a solution containing the secondary antibody (0.5% Triton X100 in 1X PBS) for 1 hour at room temperature. For mouse monoclonal antibodies, a peroxidase-conjugated goat anti-mouse IgG was used at a dilution of 1:100 in PBS. The sections were then rinsed three times in fresh 1X PBS, Then, the specimens were incubated in a solution containing 0.05% of 3,3- diaminobenzidine (DAB), 25 mg/ml nickel ammoniumsulfate and 0.03% of H2O2 in 1X
PBS (pH 7.4) for approximately 5 minutes at room temperature until the staining was visible. Subsequently, the sections were rinsed three times with 1X PBS for 10 minutes. For permounting, the sections were putted onto microscope slides, washed three times with ddH2O, and rinsed with 75%, 95%, and 100% ethanol and subsequently with Xyelene for 45
seconds. Afterwards, the slides were dried at room temperature for at least 5 minutes, droped with permount solution, and covered immediately with cover glass so that no bubbles appeared.